Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels
Author(s): ,
Ulrike Protzer
Affiliations:
German Center for Infection Research (DZIF), Munich partner site, Munich, Germany
Corresponding author. Address: Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstrasse 30, 81675 Munich, Germany. Tel.: +49 8941406886; fax: +49 8941406823.
,
Wang-Shick Ryu
Affiliations:
Department of Biochemistry, Yonsei University, Seoul, Republic of Korea
,
Jane A. McKeating
Affiliations:
Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
,
Karin Wisskirchen
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Ke Zhang
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Theresa Asen
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Romina Bester
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Daniela Stadler
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Jochen M. Wettengel
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Julia Hasreiter
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Wen-Min Chou
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
,
Anindita Chakraborty
Affiliations:
Technische Universität München, Institute for Advanced Study, Munich, Germany
Chunkyu Ko
Affiliations:
Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
EASL LiverTree™. Protzer U. Dec 1, 2018; 256733
Dr. Ulrike Protzer
Dr. Ulrike Protzer

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Graphical abstract

Graphical abstract

Studying HBV has been limited by the availability of in vitro and in vivo models. A selected HepG2 cell clone expressing NTCP supports long-term HBV infection. HBV has slow infection kinetics requiring 3 days for full establishment of infection. HBV establishes 1–9 copies of cccDNA per cell which have an estimated half-life of 40 days. cccDNA levels remain stable by intracellular genome recycling and secondary infection.

Background & Aims

Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture.

Methods

We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection.

Results

HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5–12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days.

Conclusions

After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents.

Lay summary

Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.

Keyword(s)
HBV, Hepatitis B virus, cccDNA, NTCP, Replenishment, Viral spread, Transmission, Intracellular recycling
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